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Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

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Loading different fractions of the cycling vesicle pool at hippocampal boutons. Data are from four to six coverslips per treatment, and >100 boutons were analyzed. (A) Comparison of the uptake of 4 μM FM1-43 by boutons when stimulated for varying lengths of time in either 25 (triangles), 40 (squares), or 70 mM K+ (circles). (B) Expansion of the first 2 min of A. (C) Distribution of fluorescence intensities of maximally loaded boutons. (D) Distribution of fluorescence intensities of boutons containing only three to eight labeled vesicles (25 mM K+ for 1 min). Note the absence of clear quantal peaks. (E) Destaining kinetics of maximally loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. (F) Destaining kinetics of weakly loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. Note that E and F were obtained using different settings, so the y-axes are not comparable.
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fig1: Loading different fractions of the cycling vesicle pool at hippocampal boutons. Data are from four to six coverslips per treatment, and >100 boutons were analyzed. (A) Comparison of the uptake of 4 μM FM1-43 by boutons when stimulated for varying lengths of time in either 25 (triangles), 40 (squares), or 70 mM K+ (circles). (B) Expansion of the first 2 min of A. (C) Distribution of fluorescence intensities of maximally loaded boutons. (D) Distribution of fluorescence intensities of boutons containing only three to eight labeled vesicles (25 mM K+ for 1 min). Note the absence of clear quantal peaks. (E) Destaining kinetics of maximally loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. (F) Destaining kinetics of weakly loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. Note that E and F were obtained using different settings, so the y-axes are not comparable.

Mentions: In Fig. 1 A, we show the relationship between length of time in elevated K+ concentration and FM1-43 fluorescence in synaptic boutons. The final saturating level of fluorescence was the same for three separate concentrations of K+ (25, 40, and 70 mM), although the length of stimulation required to reach that level differed. Fig. 1 B shows an expanded portion of this plot, focusing on the first 2 min in 25 and 40 mM potassium. If ∼400 fluorescence units correspond to an average of 25 vesicles, then one would predict that incubation for 1 min in 25 mM K+ would lead to the labeling of approximately five vesicles per nerve terminal. In Fig. 1 (C and D), we compare histograms of bouton intensity from individual coverslips labeled with either 70 mM K+ for 10 min or 25 mM K+ for 1 min. No pattern underlying the distribution of fluorescence intensities from the strong loading conditions was seen. However, the fluorescence intensities of boutons labeled with a weak protocol might have been expected to show quantal fluctuations in fluorescence intensity. This was not the case, for reasons described in the Discussion. Finally, we compared the rates of destaining of these two groups of boutons in response to a 70-mM K+ challenge (Fig. 1, E and F), which revealed similar destaining kinetics, suggesting that the vesicles labeled with the weak protocol were randomly mixed within the overall cycling vesicle pool.


Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

Loading different fractions of the cycling vesicle pool at hippocampal boutons. Data are from four to six coverslips per treatment, and >100 boutons were analyzed. (A) Comparison of the uptake of 4 μM FM1-43 by boutons when stimulated for varying lengths of time in either 25 (triangles), 40 (squares), or 70 mM K+ (circles). (B) Expansion of the first 2 min of A. (C) Distribution of fluorescence intensities of maximally loaded boutons. (D) Distribution of fluorescence intensities of boutons containing only three to eight labeled vesicles (25 mM K+ for 1 min). Note the absence of clear quantal peaks. (E) Destaining kinetics of maximally loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. (F) Destaining kinetics of weakly loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. Note that E and F were obtained using different settings, so the y-axes are not comparable.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171786&req=5

fig1: Loading different fractions of the cycling vesicle pool at hippocampal boutons. Data are from four to six coverslips per treatment, and >100 boutons were analyzed. (A) Comparison of the uptake of 4 μM FM1-43 by boutons when stimulated for varying lengths of time in either 25 (triangles), 40 (squares), or 70 mM K+ (circles). (B) Expansion of the first 2 min of A. (C) Distribution of fluorescence intensities of maximally loaded boutons. (D) Distribution of fluorescence intensities of boutons containing only three to eight labeled vesicles (25 mM K+ for 1 min). Note the absence of clear quantal peaks. (E) Destaining kinetics of maximally loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. (F) Destaining kinetics of weakly loaded boutons stimulated with 70 mM K+ during the time indicated by the bar. Note that E and F were obtained using different settings, so the y-axes are not comparable.
Mentions: In Fig. 1 A, we show the relationship between length of time in elevated K+ concentration and FM1-43 fluorescence in synaptic boutons. The final saturating level of fluorescence was the same for three separate concentrations of K+ (25, 40, and 70 mM), although the length of stimulation required to reach that level differed. Fig. 1 B shows an expanded portion of this plot, focusing on the first 2 min in 25 and 40 mM potassium. If ∼400 fluorescence units correspond to an average of 25 vesicles, then one would predict that incubation for 1 min in 25 mM K+ would lead to the labeling of approximately five vesicles per nerve terminal. In Fig. 1 (C and D), we compare histograms of bouton intensity from individual coverslips labeled with either 70 mM K+ for 10 min or 25 mM K+ for 1 min. No pattern underlying the distribution of fluorescence intensities from the strong loading conditions was seen. However, the fluorescence intensities of boutons labeled with a weak protocol might have been expected to show quantal fluctuations in fluorescence intensity. This was not the case, for reasons described in the Discussion. Finally, we compared the rates of destaining of these two groups of boutons in response to a 70-mM K+ challenge (Fig. 1, E and F), which revealed similar destaining kinetics, suggesting that the vesicles labeled with the weak protocol were randomly mixed within the overall cycling vesicle pool.

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

Show MeSH
Related in: MedlinePlus